Abstract The global market for `biologics', which are new-generation drugs produced using genetically engineered biological systems, is projected to reach $445 billion by 20191. One system currently being used to manufacture biologics is the baculovirus-insect cell system (BICS). The BICS offers certain advantages over more widely used mammalian cell systems, including its rapid adaptability for manufacturing different products, use of a single qualified master cell bank for all products, absence of endogenous retroviruses, and superior ability to produce multiprotein complexes. It also presents an alternative for products that are poorly produced in mammalian cell systems and enhances manufacturing capacity. However, the BICS cannot be used to manufacture glycoprotein biologics, the most common type, because it cannot produce the human-type carbohydrate side-chains, or `N-glycans' required for their clinical efficacy. GlycoBac has shown we can solve this problem using our patented technology to produce insect cell derivatives that can synthesize human-type N-glycans. So far, our `glycoengineering' efforts have focused on cell lines derived from S. frugiperda (Sf) because they are most commonly used in the BICS. However, T. ni (Tn) cell lines are also used and have the capacity for higher productivity2. Considering this, glycoengineered Tn cell lines would be highly attractive to glycoprotein biologics manufacturers. Unfortunately, glycoengineering currently available Tn lines would be useless because these lines are contaminated with a virus3 and produce immunogenic N-glycans4. These problems have thwarted GlycoBac's opportunity to leverage our technology and glycoengineer Tn cells, which would double our product catalog. Thus, we isolated a new, patent-pending Tn line that has no viral contaminants and produces no immunogenic glycans. We now propose to use this line to demonstrate the feasibility of Tn glycoengineering. First, we will knockout the endogenous fused lobes (fdl) gene, which antagonizes human-type N-glycan elongation5-7. We will do this using our new, patent-pending, lepidopteran insect-specific CRISPR-Cas9 tools8, which will provide a competitive advantage. Second, we will knock-in heterologous genes required for human-type N-glycan synthesis using our established technology9, which also will provide a competitive advantage, and characterize the product. The results will reveal if it is feasible to glycoengineer Tn cells and isolate derivatives that can produce glycoproteins with human-type N-glycans. A successful outcome will set the stage for Phase II, in which we would produce a full suite of glycoengineered Tn derivatives designed to produce recombinant glycoproteins with various clinically relevant N-glycan structures. We would extensively characterize these lines and document their safety and efficacy. These new glycoengineered insect cell lines would grow GlycoBac's business, effectively doubling our catalog, as noted above. This would enhance global manufacturing capacity for glycoprotein biologics, which have the biggest impact on human health and, therefore, enhance the biotechnology sectors' efforts to improve human health.